An evaluation of the Diamat HPLC analyser for simultaneous determination of haemoglobins A(2) and F.

ABSTRACT

The authors describe a modification of the instrumental parameters of the Diamat fully automated HPLC system for Hb A(2) assay (Bio-Rad Laboratories, Milan, Italy) in order to obtain simultaneous determination of Hb A(2) and Hb F.Hb A(2) and Hb F measurements are reproducible (within-run CV 2.6%, with Hb A(2)2.7%; 5.1%, with Hb F 1.3%) and accurate (from a comparison with two microchromatographic techniques for Hb A(2) r = 0.9639 and 0.9755; with two alkali denaturation procedures for Hb F r = 0.9990 and 0.9952; with radial immunodiffusion, r = 0.9877). Assay linearity has been confirmed for Hb A(2) concentrations between 0 and 6.0%, and for Hb F between 0 and 60%. The data obtained from the analysis of some pathological samples for Hb Bart's, Hb H, Hb J Sardegna, Hb Lepore and Hb S are in agreement with cellulose acetate electrophoresis analysis.The Hb A(2) reference intervals for normals (N = 597) and Beta-thalassemia carriers (N = 200) are respectively (95% limits) 2.02-3.27 and 3.92-5.90 in % units. Hb F values measured in normals (N = 968), in beta-thal carriers (N = 302) and in deltabeta-thal carriers (N =3) have been found to be consistent with the usual diagnostic parameters.SOME MINOR LIMITATIONS EMERGED the most relevant concerns Hb A(1c), which is overestimated with respect to a reference method (y = 1.217x + 0.16; N = 79; r = 0.9235). A probable interference from labile fractions is responsible for this Hb A(1c) inaccuracy.